G protein coupled receptors (GPCRs) constitute the largest cell-surface receptor family and at least 35% of currently prescribed drugs act on these receptor molecules. GPCR signaling is critically involved in many aspects of cardiovascular function. The magnitude and extent of GPCR signaling is determined by several governing factors including the lifetime of the receptor molecule itself. During the first period of funding, we have found that ubiquitination of the cell-surface b2 adrenergic receptor (b2AR) determines its degradation in lysosomes, thus providing an 'off switch'for attenuating cellular responses. We have identified specific enzymatic activities involved in regulating the intracellular trafficking of agonist-activated b2ARs. Thus, the RING-domain containing E3 ubiquitin ligase Mdm2 ubiquitinates the receptor associated adaptor protein b-arrestin2 and is involved in early steps of receptor internalization while the HECT- domain containing E3 ligase Nedd4 ubiquitinates the b2AR leading to receptor degradation in the lysosomes. Recruitment of both ligases to the b2AR is agonist-dependent and occurs sequentially. We have also shown that two related deubiquitinases (DUBS), USP20 and USP33 reverse this ubiquitination and prevent receptor degradation while concomitantly promoting receptor recycling to the plasma membrane. The central hypothesis for the proposed work in this competing continuation application is: "b-adrenergic signaling is intimately linked to trafficking pathways and involves dynamic regulation by distinct E3 ligases and deubiquitinases". By using aortic vascular smooth muscle cells and neonatal ventricular myocytes as cellular model systems, RNAi and knockout mice, we will define the impact of ubiquitination/deubiquitination dynamics on bAR responsiveness in the cardiovascular system.
The specific aims are: 1) To determine the effects of lysosomal trafficking in regulating bAR signaling, 2) To elucidate the molecular mechanisms that define the recruitment and/or activation of deubiquitinases during bAR resensitization and 3) To elucidate the mechanistic role of Mdm2 in bAR signaling in the heart. The long-term goal of this project is to understand the molecular mechanisms that integrate G protein-coupled receptor trafficking and signaling, which could play a critical role in balancing physiological responsiveness.

Public Health Relevance

b adrenergic receptors (b1 and b2 ARs) are expressed in the heart and are important for the contractility of heart muscles, especially during stress and exercising. b2ARs also regulate the relaxation of smooth muscle cells that line the wall of blood vessels in our body. The proposed work will elucidate how cell surface expression and function of the bARs are maintained and the knowledge gained will help the development of novel therapeutics, which could be beneficial in the treatment of heart failure and blood pressure disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Wong, Renee P
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Duke University
Internal Medicine/Medicine
Schools of Medicine
United States
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Shenoy, Sudha K (2014) Arrestin interaction with E3 ubiquitin ligases and deubiquitinases: functional and therapeutic implications. Handb Exp Pharmacol 219:187-203
Shenoy, Sudha K (2014) Deubiquitinases and their emerging roles in ?-arrestin-mediated signaling. Methods Enzymol 535:351-70
Han, Sang-Oh; Kommaddi, Reddy P; Shenoy, Sudha K (2013) Distinct roles for ?-arrestin2 and arrestin-domain-containing proteins in ?2 adrenergic receptor trafficking. EMBO Rep 14:164-71
Shenoy, S K; Han, S; Zhao, Y L et al. (2012) ýý-arrestin1 mediates metastatic growth of breast cancer cells by facilitating HIF-1-dependent VEGF expression. Oncogene 31:282-92
Wu, Jiao-Hui; Zhang, Lisheng; Fanaroff, Alexander C et al. (2012) G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors. Arterioscler Thromb Vasc Biol 32:308-16
Shenoy, Sudha K; Lefkowitz, Robert J (2011) *-Arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol Sci 32:521-33
Shenoy, Sudha K (2011) Visualizing G protein-coupled receptor signalsomes using confocal immunofluorescence microscopy. Methods Mol Biol 756:333-42
Schulte, Gunnar; Shenoy, Sudha K (2011) ýý-Arrestin and dishevelled coordinate biased signaling. Proc Natl Acad Sci U S A 108:19839-40
Xiao, Kunhong; Shenoy, Sudha K (2011) Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains. J Biol Chem 286:12785-95
Cai, Xinjiang; Wu, Jiao-Hui; Exum, Sabrina T et al. (2009) Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis. Mol Pharmacol 75:626-36

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